# MHC1-TIP enables single-tube multimodal immunopeptidome profiling and uncovers intratumoral heterogeneity in antigen presentation

## TL;DR

A new method called MHC1-TIP allows detailed analysis of immune-related proteins from small tumor samples, revealing hidden differences in antigen presentation.

## Contribution

MHC1-TIP is a scalable, low-input, single-tube workflow for robust immunopeptidome profiling with multimodal compatibility.

## Key findings

- MHC1-TIP enables immunopeptidome profiling from sub-milligram tumor fragments and patient-derived organoids.
- Intratumoral heterogeneity in antigen presentation was found to be poorly correlated with proteomic expression in renal cell carcinoma.
- The method supports integrated analysis of proteome and immunopeptidome from the same sample.

## Abstract

Profiling antigens presented on MHC class I molecules on the cell surface is essential to identify candidate antigens for targeted and personalized immunotherapies. However, mass spectrometry-based immunopeptidomics has traditionally been limited by high input requirements, extensive sample manipulation, and expensive reagents. To overcome these challenges, we developed MHC1-TIP: a scalable, single-tube and cost-effective workflow to enable robust MHC-I ligandome recovery from cell lines, patient-derived organoids, and sub-milligram amounts of ex-vivo tumour fragments. Moreover, MHC1-TIP also preserves compatibility with additional omics profiling technologies and we demonstrate its capacity for quantitative and multimodal profiling of the proteome and immunopeptidome from the same sample to enable integrated analyses of protein expression and antigen presentation. Application of MHC1-TIP to primary renal cell carcinoma fragments revealed extensive intratumoral heterogeneity in antigen presentation that was poorly correlated with source protein expression. MHC1-TIP represents a broadly applicable and sensitive approach for low-input, multimodal immunopeptidomics with clinical and translational relevance.

A novel low-input, single-tube immunopeptidomics method uncovers widespread intratumor heterogeneity in MHC-I antigen presentation that is largely uncoupled from proteomic heterogeneity.